Salmonella contamination of food products, caused by Salmonella enterica, which infects humans, cattle, and poultry, is a serious public health problem, resulting in product recalls and illness. Moreover, Salmonella has been used successfully as a bioterrorism agent and is listed as a category B pathogen by the NIH/NIAID. Recently, Salmonella strains resistant to heretofore efficacious antibiotics have emerged. Thus, it is of high importance to develop novel therapeutic agents directed at Salmonella. The development of an antimicrobial that targets a novel molecular pathway would prove very useful in combating such resistant strains. The ubiquitin proteasome pathway has excellent potential in this regard. The novel virulence factor SseL, which plays a key role in both Salmonella virulence and macrophage killing during infection (expression of SseL enhances virulence and macrophage killing), is known to be translocated into the host cell and function as a deubiquitinase, a class of protease that deconjugates ubiquitin from various proteins, thereby helping to regulate various essential cell functions. It is therefore proposed to develop a novel therapeutic capable of treating Salmonella pathogenesis based on the inhibition of SseL function. Progenra has developed a highly sensitive assay platform for measuring deubiquinase activity; it is based on the N-terminal fusion to ubiquitin of a reporter enzyme (phospholipase A2) that requires a free N terminus for activity, which is liberated upon deubiquitinase-catalysed cleavage of the ubiquitin fusion. This platform will be applied in Phase I to configure a high throughput screen for inhibitors of SseL activity. A pilot screen will be conducted using a protease directed library of compounds to verify that the assay is functioning properly. Positives in the assay will be tested in a cell-based assay for macrophage killing secondary to S. enterica infection. In Phase II, natural product extracts and compound collections will be screened for inhibitors of SseL, and leads will be identified, optimized, and developed as S. enterica antimicrobials. The Category B pathogen Salmonella enterica, which infects humans, cattle, and poultry, is a serious public health problem, resulting in product recalls and illness. This organism is considered a potential bioterrorism threat by the NIH. Recently, Salmonella strains resistant to antibiotics have emerged, stressing the importance of developing new medicines to treat infection with such resistant strains. Recently a new target has been identified, inhibitors of which are predicted to be good candidates for new drugs to fight Salmonella. Progenra has developed a highly sensitive assay to measure the activity of this target and thereby help to identify inhibitors for testing in humans. In Phase I, a compound collection will be screened for inhibitors of the target, and the best inhibitors found will be tested further for activity against Salmonella in macrophage cells. In Phase II, additional screening will be conducted and compounds identified will be advanced to the stage at which clinical candidates can be selected from among them. [unreadable] [unreadable] [unreadable]